Chromosomal functions are regulated by dynamic modifications of their structural proteins. Acetylation and deacetylation are well-established modifications that affect how histones and other chromosomal proteins influence transcription, recombination, replication and repair of damage. A distinct, newly defined activity is SUMO-targeted ubiquitin ligation (STUbL), catalyzed by proteins previously identified for their roles in genome stability and in response to DNA. The goals of the research are to define the mechanisms by which chromatin deacetylation and STUbL together yield optimal transcriptional silencing, genome stability and growth regulation. The proposed research builds on the lab's recent discovery that the Sir2 deacetylase is physically and functionally linked to a STUbL activity catalyzed by the Slx5-Slx8 complex. The project will be accomplished through three aims. In the first aim, genetic and biochemical studies will test the hypothesis that a second predicted STUbL protein functions in parallel to SLX5-SLX8 to promote optimal structure and function through STUbL activity. Transcriptional silencing defects of mutants will be characterized through molecular and genetic approaches, including genetic analysis and chromatin immunoprecipitation (ChIP). In the second aim, the genomic and subnuclear localization of STUbL components will be evaluated. Genomic and cell biological experiments will test if STUbLs occupy silent chromatin and define a distinct genomic binding pattern and subnuclear compartment(s). The third aim will define chromatin-specific substrates of STUbL activity through biochemical approaches. A combination of candidate substrate and proteomic analyses will be used. Potential substrates will be independently validated through additional biochemical and molecular genetic approaches. It will be determined if Sir2 deacetylase activity influences STUbL activity or substrate specificity. Together, the results from these three aims will establish the key mechanisms, substrates, and genomic targets of STUbL that are critical for chromatin function. PUBLIC HEALTH RELEVANCE: The STUbL enzymes that are the focus of this study affect critical processes such as cell growth, aging, metabolism, and response to DNA damage. In human cells, a STUbL was recently shown to be responsible for the therapeutic benefits in treatments of acute promyelocytic leukemia, yet many unanswered questions remain about this newly identified family of enzymes. Understanding their cellular roles, substrates and regulation will ultimately provide insight into basic processes that define human development and disease, and may be particularly relevant to mechanisms of healthy aging and cancer.